MLL/HRX/ALL1 encodes a 3,969 amino acid nuclear protein bearing multiple conserved domains with assigned activities including: an N terminus with three AT-hook motifs that bind AT rich DNA segments (Zeleznik-Le et al., (1994) Proc. Natl. Acad. Sci. USA, 91:10610-10614), a DNA methyl transferase homology domain that represses transcription (Xia et al., (2003) Proc. Natl. Acad. Sci. USA), four PHD fingers that mediate protein-protein interactions (Fair et al., (2001) Mol. Cell. Biol., 21:3589-3597), a transactivation domain that interacts with CBP (Ernst et al., (2001) Mol. Cell. Biol., 21:2249-2258), and a C-terminal SET domain with histone H3 lysine 4 methyl transferase activity (Milne et al., (2002) Mol. Cell., Vol. 10:1107-1117; Nakamura et al., (2002) Mol. Cell., 10:1119-1128) (FIG. 1A). MLL and its Drosophila homologue trithorax are required for maintaining proper Hox and homeotic gene expression patterns, respectively (Breen and Harte, (1993) Development, 117:119-134; Yu et al., (1998) Proc. Natl. Acad. Sci. USA, 95:10632-10636).
Chromosome translocations characteristically found in human infant leukemia disrupt MLL (11q23), generating chimeric proteins between the MLL N-terminus and multiple translocation partners that vary substantially (Ayton and Cleary, Oncogene, (2001) 20:5695-5707; Domer et al., (1993) Proc. Natl. Acad. Sci. USA, 90:7884-7888; Downing and Look, (1996) Cancer Treat. Res., 84:79-92; Gu et al., (1992) Cell, 71:701-708; Thirman et al., (1993) New England Journal of Medicine, 329:909-914; Tkachuk et al., (1992) Cell, 71:691-700). Mice carrying engineered Mll translocations develop leukemia (Corral et al., (1996) Cell, 85:853-851; Forster et al., (2003) New England Journal of Medicine, 326:800-806). Gene expression profiles of infant leukemias bearing MLL translocations identified a characteristic gene expression profile that distinguishes this poor prognosis leukemia from other leukemias (Armstrong et al., (2002) Nat. Genet., 30:41-47; Yeoh et al., (2002) Cancer Cell, 1: 133-143). Among the upregulated genes were some recognized targets of MLL including select HOX genes. Deregulated expression of HOX genes typifies certain malignancies (Buske and Humphries, (2002) Int. J. of Hematol., 71:391-398; Cillo et al., (2001) Int. J. Hematol., 71:161-169; Dash and Gilliland, (2001) Best Pract. Res. Clin. Haematol., 14:49-64).
Recently, we and others demonstrated that MLL is normally processed at two cleavage sites, CS1 (D/GADD) and CS2 (D/GVDD), and that mutation of both sites abolishes the proteolysis (Hsieh et al., (2003) Mol. Cell. Biol., 23:186-194; Yokoyama et al., (2002) Blood, 100:3710-3718) (FIG. 1B). The sequence of the cleavage site is highly conserved in MLL homologues from flies to mammals. MLL cleavage generates N-terminal p320 (N320) and C-terminal p180 (C180) fragments, which heterodimerize to form a stable complex that localizes to a subnuclear compartment. Within this complex, the FYRN domain of N320 directly interacts with the FYRC and SET domains of C180. This dynamic post-cleavage association confers stability to N320 and correct nuclear sublocalization of the MLL complex for proper target gene expression (Hsieh et al., (2003) Mol. Cell. Biol., 23:186-194).
Site-specific proteolysis is essential in many important biological pathways including the sequential activation of blood coagulation factors (Furie and Furie, (1992) New England Journal of Medicine, 326:800-806), cholesterol-gauged liberation of SREBP from the ER (Brown et al., (2000) Cell, 100:391-398), ligand-activated cleavage and subsequent release of the intracellular domain of Notch (Brown et al., (2000) Cell, 100:391-398), maturation of the hedgehog signaling molecule (Ye and Fortini, Semin. (2000) Cell Dev. Biol., 11:211-221), separation of HCF-1 for proper cell cycle regulation (Wilson et al., (1995) Genes. Dev., 9:2445-2458), and activation of caspases and their subsequent cleavage of death substrates during apoptosis (Thornberry and Lazebnik, (1998) Science, 281:1312-1316). Identification and characterization of the responsible proteases has not only proven critical to understanding such biologic processes but also for developing targeted therapeutics for diseases involving specific pathways.